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Lipid bilayer membranes are indispensable parts of cellular architecture. One of the integral properties of bilayer membranes is the environmental heterogeneity over a wide range of spatiotemporal scales. The environmental heterogeneity is a manifestation of the dynamic and compositional anisotropy in the plane of the membrane as well as along the bilayer normal. Fluorescence lifetime distribution analysis provides a spectroscopic tool to quantitatively characterize such heterogeneities. The review discusses recent applications of fluorescence lifetime distribution analysis utilizing the maximum entropy method to characterize horizontal and vertical heterogeneities in membranes.

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Electroanalytic, photometric or fluorometric methods may provide information about the presence of proteolysis in a related sample, but they cannot accurately identify which protease belongs to the proteolytic activity. In other words, they cannot distinguish the proteases according to the differences in their activities. Previous studies on the chymotrypsin and trypsin have shown that the activities of proteases can be distinguished from each other by fluorescence lifetime distributions. In this study, it is aimed to show the sensitivity of the distributional model on the proteolytic activities of the two proteases. For this purpose, the proteolytic activities have been reduced by making covalent conjugation with polyacrylic acid (PAAc) and the effects of the low activities were examined on Bovine Serum Albumin (BSA) which excimer emission character was incorporated into its structure by modification with N-(1-pyrenyl)maleimide. The time-resolved spectrofluorometer was used to collect fluorescence decay data at λ(excimer) = 464 nm, which were analyzed by using Exponential Series Method (ESM) to obtain the changes of lifetime distributions. The results showed a significant decline in the activities. Despite the very low activities, the changes of fluorescence lifetime distributions exhibited remarkable specific differences that proved the sensitivity of ESM analysis.

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Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.

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Tryptophan is the most often investigated intrinsic fluorophore due to its abundance in proteins and its sensitivity to different environmental conditions. Fluorescence quenching is a powerful method to study proteins and acrylamide is a frequently applied quencher in these investigations. Quenching experiments are sometimes distorted by the undesired protein-quencher interactions that can result in a misinterpretation of the results. Here we focused on the identification of the possible side-effects of acrylamide applying fluorescence lifetime measurements. To provide reference data for protein denaturation the fluorescence parameters were also recorded in the presence of different concentrations of guanidine hydrochloride. In circular dichroism experiments we characterized directly the acrylamide effect on the tertiary structure of the proteins. According to the obtained data in experiments with seven tryptophan-containing proteins the full width at half maximum (FWHM) of the fluorescence lifetime distribution is an appropriate parameter to monitor the undesired effects of acrylamide on the proteins.

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Biological membranes display considerable anisotropy due to differences in composition, physical characteristics, and packing of membrane components. In this Letter, we have demonstrated the environmental heterogeneity along the bilayer normal in a depth-dependent manner using a number of anthroyloxy fatty acid probes. We employed fluorescence lifetime distribution analysis utilizing the maximum entropy method (MEM) to assess heterogeneity. Our results show that the fluorescence lifetime heterogeneity varies considerably depending on fluorophore location along the membrane normal (depth), and it is the result of the anisotropic environmental heterogeneity along the bilayer normal. Environmental heterogeneity is reduced as the reporter group is moved from the membrane interface to a deeper hydrocarbon region. To the best of our knowledge, our results constitute the first experimental demonstration of anisotropic heterogeneity in bilayers. We conclude that such graded environmental heterogeneity represents an intrinsic characteristics of the membrane bilayer and envisage that it has a role in the conformation and orientation of membrane proteins and their function.